The present invention relates to new bacterial receptor structures originating from natural bacterial receptor structures which have been modified in regard to amino acid residues involved in the original interaction function, whereby said original interaction function has been substantially inhibited and replaced by a modified interaction function directed to a desired interaction partner.
Several bacteria known to invade mammals have evolved surface proteins capable of binding to a variety of substances including host specific carbohydrates and proteins. Several such receptors from Gram-positive bacterial pathogens have been isolated and characterized in detail as will be shown below. Most well-characterized are the Fc receptors, named after the capability of binding to the constant Fc part of IgG. Based on binding experiments to IgG from various mammalian sources, and subclasses thereof, Fc receptors have been divided into six types I-VI. The receptor from S.aureus, protein A [SPA], defining the type I receptor, has been the subject of immense studies.
SPA binds IgG from most mammalian species, including man. Of the four subclasses of human IgG, SPA binds to IgG1, and IgG4 but shows very weak or no interaction with IgG3 [Eliasson, M. et al, 1989 J.Biol.Chem. 9:4323-4327]. This pseudoimmune reaction has been used for more than 20 years for the purification and detection of antibodies in diagnostic, research and therapeutic applications. Cloning, sequencing and Escherichia coli expression of defined fragments of the SPA gene revealed a highly repetitive organization, with five IgG binding domains [E-D-A-B-C], a cell wall spanning region and membrane anchoring sequence [XM] [Uhlxc3xa9n, et al, 1984 J.Biol.Chem. 259:1695-1702; Moks, T. et al, 1986 Eur.J.Biochem. 156:637-643]. A vast number of plasmid vectors have been constructed, allowing gene fusions to different fragments of the gene for the purpose of fusion protein production in different hosts [Nilsson B. and Abrahmsxc3xa9n, L. 1990 Meth.Enz. 185:144-161] (FIG. 2a).
The structure for a complex between human Fc [IgG1] and a single domain [B] of SPA has been determined by X-ray crystallography at a resolution of 2.8 xc3x85 [Deisenhofer, J. et al 1981 Biochemistry 20:2361-2370]. Based on this structure and additional information from NMR experiments, the B domain can be viewed as a compact structure consisting of three anti-parallel xcex1-helices connected with loops. In the Fc binding, which is of both electrostatic and hydrophobic nature, only side chains of residues from helices 1 and 2 are involves, whilst the third helix is not participating in the binding. Based on this domain B, a synthetic IgG-binding domain [Z] [Nilsson, B. et al 1987 Prot,Eng. 1:107-113] has been constructed, suitable as fusion partner for the production of recombinant proteins which allows purification by IgG affinity chromatography. The high solubility and the stable structure of the Z domain has been utilized for production, purification and renaturation of a large number of recombinant proteins. [Josephsson, S. and Bishop, R. Trends Biotechnol. 6:218-224; Samuelsson, E. et al 1991 Bio.Technol. 9:363-366]
Streptococcal strains of serological groups C and G display a binding repertoire for mammalian IgGs, including human IgG3, which is even broader than for the type I receptor. The name protein G was suggested for this type III receptor from group G streptococci. In 1986 Olsson and co-workers reported on the cloning and sequencing of the gene from the serological group G streptococci [G148] [Guss, B. et al, 1987 EMBO J. 5:1567-1575; Olsson, A. et al, 1987 Eur.J.Biochem. 168:319-324]. In analogy with SPA is SPG a repetitively arranged molecule, comprising an IgG-binding region of three homologous domains [C1,C2,C3], spaced by smaller D-regions (FIG. 2A). Compared to SPA, SPG displays a different binding spectra for immunoglobulins from different species and subclasses thereof. The IgG binding domains of protein G are now widely used as an immunological tool, i.e. in the affinity purification of monoclonal antibodies. Production of subfragments constructed by DNA-technology, have shown that an individual C-region is sufficient for full IgG-binding. Recently, the structure for a complex between the C1-domain from SPG and human Fc was determined with X-ray crystallography (FIG. 2B). This shows that SPG binds to the CH2-CH3 interface but at a different site compared to SPA. The binding is mainly of electrostatic nature which is in contrast to the large contribution of hydrophobic forces seen for the SPA-Fc interaction. Moreover, the 3-D structure of C1 differs from the X structure in that it is built up by two xcex2-sheets connected by an xcex1-helix [xcex2xcex2-xcex1-xcex2xcex2]. The residues of C1 which according to the structure are involved in the binding, corresponds to the xcex1-helix, the loop and the following xcex2-sheet.
An additional activity of SPG is the capability to bind serum albumin. The binding strength is species dependent, and among the tested samples. SPG binds strongest to serum albumin from rat, man and mouse. Production and binding studies of subfragments of SPG shows that the two binding activities are structurally separated and that the serum albumin binding function is located at the repetitive A-B region [Nygren et al 1990 Eur.J.Biochem. 193:143-148]. This region has been used for several biotechnological purposes. Recombinant proteins have been produced as fusions to the region which enables the purification by affinity chromatography, where human serum albumin most frequently has been used as immobilized ligand. Proteins found to be proteolytically sensitive have been produced as xe2x80x9cdual affinity fusionsxe2x80x9d in which they are flanked by two different affinity tails derived from SPA and SPG, respectively. Purification schemes employing both the N- and C-terminal are thus possible which ensures the recovery of an intact target protein [Hammarberg et al 1989 Proc.Natl.Acad.Sciences USA 86:4367-4371]. The strong and specific binding to serum albumin has also been used for the in vivo stabilization of therapeutic proteins.
Through complex formation with the very long-lived serum albumin, the receptor is carried in the circulation (macque apes) with a half-life which is close to the half-life for serum albumin itself. Studies in mice with the for HIV/AIDS therapy interesting, but rapidly cleared T-cell receptor CD4, showed that it was substantially stabilized when fused to the serum albumin binding region, when compared with an unfused control protein [Nygren et al 1991 Vaccines 91 Cold Spring Harbor Press 363-368]. The slow clearance can probably be explained by the complex formation with serum albumin which circumvents elimination by the liver and excretion in the kidney.
In order to determine the minimal extension required for maintained binding to serum albumin, several smaller fragments of the A-B region have been produced and analyzed. The smallest fragment so far with serum albumin binding activity is a 46 residue fragment [xe2x80x9cB2A3xe2x80x9d] comprising region A3 flanked by 13 and 9 residues, respectively, from regions B2 and S.
Based on homology and binding studies of other partial fragments SPG is regarded to be trivalent with regard to binding to serum albumin. Similar to the monovalent IgG-binding domains Z and C1 B2A3 is relatively small and shows high solubility and stability and is therefore a suitable candidate for modification.
The present invention has for its main purpose to provide new bacterial receptor structures by modifying natural bacterial receptors in regard to their original interaction functions to result in new structures having modified interaction functions.
Another object of the invention is to provide artificial bacterial receptor structures which are stable and more resistant to various conditions, such as increased temperatures.
Yet another object of the invention is to provide artificial bacterial receptor structures, the interaction functions of which have been modified to direct same to other desired interaction partners.
With these and other objects that will be clear from the following disclosure in mind the invention provides for novel proteins obtainable by mutagenesis of surface-exposed amino acids of domains of natural bacterial receptors said proteins being obtained without substantial loss of basic structure and stability of said natural bacterial receptors. Said proteins have preferably been selected from a protein library embodying a repertoire of said novel proteins. In such novel bacterial receptor structures, at least one amino acid residue involved in the interaction fuction of the original bacterial receptor has been made subject to substitution by another amino acid residue so as to result in substantial loss of the original interaction capacity with a modified interaction capacity being created, said substitution being made without substantial loss of basic structure and stability of the original bacterial receptor.
It is preferred that said bacterial structures originate from Gram-positive bacteria. Among such bacteria there may be mentioned Staphylococcus aureus, Streptococcus pyogenes [group A], Streptococcus group C,G,L, bovine group G streptococci, Streptococcus zooepidemicus [group C], Streptococcus zooepidemicus S212, Streptococcus pyogenes [group A], streptococci groups A,C,G, Peptostreptococcus magnus, Streptococcus agalactiae [group B].
Of special interest are thermophilic bacteria evolved to persist in environments of elevated temperatures. Receptors from species like e.g. Bacillus stearothermophilus, Thermus aquaticus, Thermococcus litoralis and Pyrococcus have the potential of being naturally exceptionally stable, thus suitable for providing structural frameworks for protein engineering according to the invention.
It is particularly preferred to use as a starting material for the modification of the interaction function bacterial receptor structures originating from staphylococcal protein A or streptococcal protein G.
Among preferred receptors there may be mentioned bacterial receptors originating from Fc[IgG]receptor type I, type II, type III, type IV, type V and type VI, fibronectin receptor, M protein, plasmin receptor, collagen receptor, fibrinogen receptor or protein L [K light chains], protein H [human IgG], protein B [Human IgA,A1], protein Arp [human IgA].
Particularly preferred bacterial receptors originate from the Fc[IgG]receptor type I of staphylococcal protein A or the serum albumin receptor of streptococcal protein G.
In order to maintain stability and the properties of the original bacterial receptor structure it is preferred in accordance with the present invention that the substitution involving amino acid residues taking part in the interaction function of the original bacterial receptor does not involve more than about 50% of the amino acid residues of the original bacterial receptor. It is particularly preferred that not more than about 25% of the amino acid residues of the original bacterial receptor are made subject to substitution.
In regard to the original bacterial receptor structures selected from modification of their interaction functions it is particularly preferred to use receptors originating from the IgG-binding domains Z, Cl, and the serum albumin binding domains B2A3.
In order to maintain as far as possible the stability and properties of the original receptor structure subject to modification in accordance with the present invention it is preferred that substitution thereof involves not more than substantially all of the amino acid residues taking part in the interaction function of the original bacterial receptor.
In order to obtain favourable properties concerning stability and resistance to various conditions it is preferred that the bacterial receptor according to the present invention is comprised of not more than about 100 amino acid residues. It is known from scientific reports that proteins of a relatively small size are fairly resistant to increased temperatures and also to low pH and certain chemicals. For details concerning temperature resistance c.f. the article by Alexander et al. in Biochemistry 1992, 31, pp 3597-3603.
With regard to the modification of the natural bacterial receptor structure it is preferred to perform the substitution thereof by resorting to a genetic engineering, such as site-directed mutagenesis.
With regard to the interaction partner of the modified natural bacterial receptor a multitude of substances are conceivable, such as proteins, lipids, carbohydrates and inorganic substances. Among carbohydrates examples are blood group determinants and pathogen specific oligosaccharides.
In regard to proteins conceivable interaction partners are IGF-I, IGF-II, hGH, Factor VIII, insulin and apolipoprotein and their respective receptors as interaction partners. Furthermore, by selecting new receptor variants with specificity for different folding forms of proteins, affinity resins or analytical tools to facilitate the isolation of correctly folded molecules can be produced. Further examples are viral coat proteins, bacterial antigens, biotin and cell markers, such as CD 34 and CD 4.
Although the present invention is applicable to a variety of natural bacterial receptors the following illustration of the invention more in detail will be directed to the use of the IgG-binding domains Z, Cl and B2A3. The concept of the present invention residing in the use of artificial bacterial receptors based on the natural structures of naturally occurring bacterial receptors is associated with several advantages. Thus, the invention makes it possible to use robust and stable, highly soluble and secretion competent receptors. This is in contrast to previous techniques based on the use of polyclonals and monoclonals, such as for diagnostic purposes, which are not very stable in connection with storage, varying conditions, such as varying temperatures etc. Furthermore, the invention makes it possible to modify natural bacterial receptors to obtain desired interaction capacities for specific purposes.
For the selection of such functional variants in a large repertoire, a powerful selection system must be employed. Recent developments in this field offer alternative methods. One of the most important tools for protein engineering that has emerged during the last years is the phage display of proteins. By recombinant DNA techniques, single phage particles can be prepared which on their surface carries a protein fused to a phage-coat protein. By panning from a large pool of phages bearing different proteins, or variants of a specific protein, specific phage clones can be sorted out, which displays a certain binding characteristic [WO92/20791 to Winter et al]. Since the phage particle contains packed DNA encoding the phage protein components, a coupling between the specific variant of the displayed protein and the corresponding genetic information is obtained. Using this technique, typically 109 phage clones can simultaneously be generated and subjected to panning for screening of desired characteristics. The phage display technique can be used for selection of both small peptides as well as more complicated proteins such as antibodies, receptors and hormones. For selection of proteins which cannot be secreted, which is a prerequisite for phage display, intracellular systems have been developed in which the library of proteins are fused to a repressor protein with affinity for a specific plasmid-borne operator region resulting in a coupling between the specific protein variant and the plasmid that encoded it. An alternative to the phages as bearere of protein libraries be to use bacterial cells. Recently, display of recombinant proteins on the surface of Staphylococcus xylosus based on fusions to the cell-wall anchoring domain was demonstrated, which opens the possibility of display also of repertoires of proteins for affinity selection of specific variants [Hansson, M. et al 1992 J.Bacteriology 174:4239-4245]. Furthermore, by structure modelling using computer graphic simulations, predictions of the binding and function of altered variants of a protein can theoretically be done before the construction of the gene encoding the protein.
As indicated above the present invention describes the construction of novel proteins based on the mutagenesis of surface exposed amino acids of domains derived from bacterial receptors. These artificial bacterial receptors can be selected for different applications using a phage display system. The benefits from using bacterial receptors as structural frameworks are several. They have evolved to express a binding function without disturbing the overall structure. They are naturally highly soluble, robust to unphysiological conditions, such as pH and heat, folding efficient and are in addition secretion competent.
The invention finds use in several different areas.
The introductory part of the above-identified patent specification WO92/20791 gives an excellent survey on antibodies and their structure. Reference is particularly made to page 1 thereof.
The bacterial receptors SPA and SPG have been widely used in antibody technology for detection and purification of antibodies from e.g. hybridom supernatants and ascites fluids. However, not all antibodies are recognized by these receptors, depending on species and subclass. For the smaller subfragments of antibodies (FIG. 4), SPA and SPG show a limited binding, and efficient tools for general purification schemes are lacking. However, from a repertoire of mutant receptors including SPA and SPG, forms displaying a broader affinity for antibodies and subfragments thereof can potentially be selected.
The complex structural organization of antibodies has a number of consequences for their use in different applications as well for the production of recombinant derivatives. For use in immunosorbents, the arrangement of subunits connected by disulphide bonds can lead to a leakage of released heavy and light chains from columns. The requirement of successful docking of the two subunits contributing to the antigen binding site makes the production in bacteria of small subfragments with a low association difficult. The folding of the antibody is dependent on the formation of intra- and inter chain disulphidebonds, which are not able to form in the intracellular environment of bacterial cells. High-level intracellular expression systems for recombinant antibodies leads to the formation of inclusion bodies, which have to be renatured to obtain biological activity. These limitations make it worthwhile to search for alternatives for use as protein domains capable of specific binding, to replace antibodies in a vast number of applications.
The CDR regions forming the antigen bidning part of an antibody forms a total surface available for the antigen of approximately 800 xc3x852, with typical 10-20 residues from the antibody involved in the binding. Using the structure of the complex determined by X-ray crystallography between an individual domain B of SPA and human fc[IgGI] as a starting point about 15 amino acids of the said domain involved in this binding can be determined or postulated. The binding surface of about 600 xc3x852 is of the same order of magnitude as between an antibody and its antigen. By arbitrary in vitro mutagenesis of these positions simultaneously there is obtained a large library of Z variants with modified functional properties. In view of the fact that the regions of the Z domain constituting the very stabile so called three-helix bundle is maintained in its native form spectra of proteins are generated which could be considered as xe2x80x9cartificial antibodiesxe2x80x9d and which have the expected high solubility and excellent folding properties capable of binding to a large number of new ligands. Fusions of these artificial receptors to constant regions can be constructed to recruite effector functions, such as complement binding or triggering of ADCC (antibody dependent cellular cytotoxicity).
There are several potential advantages of utilizing the SPA structure [Z] as a starting point for such xe2x80x9cartificial antibodiesxe2x80x9d or artificial bacterial receptors. For a period of about 10 years a large number of proteins have been produced as fusions to SPA, where one has utilized the unique properties of the fusion partner in expression, refolding and purification. In these applications the Z domain has been found to be extremely soluble, stable against proteases, easy to produce in large amounts and foldable to a correct structure also intracellularly in Escherichia coli (no cysteins). Immunoglobulins (Ig:s) are substantially tetramers built up from so called xcex2-sheet structures which stabilize the orientation of the antigen-binding loops which in turn consist of continuous peptide sequences. This is to be compared to the monomeric Z domain built up from so called three-helix bundle consisting of three closely packed xcex1-helix structures, where the Fc-binding amino acids are found discontinuously in the sequence but in the folded protein are positioned on one and the same binding surface. This difference with regard to the structural elements contributing to the formation of the binding surface enables new possible conformations which cannot be obtained in natural antibodies. The ability of Z to be folded to the native structure also under conditions prevailing in the site of cytoplasma opens the possibility of using also derivatives thereof clinically. Genes coding for artificial antibodies with for example virus-neutralizing capacity can be distributed to cells through so called gene therapy resulting in interrupting the infection at an early stage.
From structure data for the complex between an individual Ig-binding domain [C1] of SPG and human Fc the binding surface can be studied. The binding which is essentially of an electrostatic nature involves side chains from amino acids from the xcex1-helix as well as from the subsequent xcex2-sheet [#3]. These differences in structure compared to the Z domain makes it useful also to create a library of C1 variants to investigate whether differences in binding patterns for artificial antibodies can be observed depending on the different conditions as regards the topology of the binding surface. Repertoires based on the structures of these and other receptors therefore offer different possibilities in the creation of artificial forms with novel functions.
When producing recombinant proteins the purification of the product is frequently a major problem. By expressing the target protein as a fusion to a so called affinity tail the hybrid product can effectively and selectively be recovered from the cell lysate or in certain cases from the culture medium by passage through a column containing an immobilized ligand. Several such gene fusion systems have been described which are based on the interaction of a certain protein with a ligand. For industrial applications it is often desirable to clean effectively the columns between the runs to satisfy purity requirements by authorities. Depending on the nature of proteins the relatively harsh conditions (NaOH, acids, heat) often used for organic or physical matrises, for example in ion exchange chromatography and gel filtration, can normally not be used. Here the use of new ligands based on stable structures originating from bacterial receptors are of great importance. In this connection the Z domain from SPA is an excellent example since said domain can be subjected to such difficult conditions as a pH of 1 or heating to 80xc2x0 C. without denaturing non-reversibly (see Example 2 below). From the library of for example Z variants interesting protein products can be selected for use immobilized on a solid phase for affinity chromatography. These protein ligands are resistant to effective purification conditions and are therefore useful repetitively on a large scale. In traditional immuno affinity chromatography where immobilized monoclonal antibodies are used for the selective purification of a certain product there are problems with leakage from the column of subunits (heavy and light chains) of the antibody since it consists of four polypeptide chains linked by cystein bridges. Since the artificial bacterial receptors consist only of one polypeptide chain this problem will be avoided. One particular area of interest is selection for binding to carbohydrates. Lectins, nature""s binders to this large and important group of biomolecules, have been found to be difficult to purify and have limited stability. Since the generation of antibodies against carbohydrates has been found to be quite complicated a selection of new artificial lectins will be of great importance to research, diagnostics and therapy.
In the production of recombinant proteins in bacterial hosts precipitates of the gene product are frequently formed, so called inclusion bodies. In order to obtain a native structure of the protein this must be subjected to refolding in vitro. A limitation in such process one is often confronted with is the fact that a great part of the material precipitates in the procedure which results in low yields. By producing the protein with an extension in the form of either a short hydrofilic peptide or an easily soluble complete domain [Samuelsson, E. et al 1991 Bio/Technol. 9:363-366] substantially higher concentrations of the protein will be obtained without precipitation taking place during renaturation. For example the high solubility of the said domain enables the use of increased solubility of proteins in either refolding from inclusion bodies or in so called reshuffling of disulphide bridges. From libraries of artificial receptors new forms can be selected having improved propeties to facilitate and even make refolding of recombinant proteins possible (cis-acting chaperones).
Recently a new unit operation for the purification of recombinant proteins based on ion exchange chromatography in so called expanded bed has been described (Hansson et al., 1994, Single-step recovery of a secreted recombinant protein by expanded bed adsorption, Biotechnol. (NY) 12: 285-288). In this connection there is used a difference in isoelectric point between the target protein and the proteins of the host cell for selective enrichment on a positively charged ion exchange matrix. By fusion to the acid Z domain (pI 4.7) the ion exchange step can take place at a pH, at which the majority of the contaminants were of the opposite charge compared to the fusion protein. By constructing libraries of bacterial receptors where a selection of amino acids have been replaced by the acid amino acids aspartate and glutamate also very acid and solubility increasing domains can be produced for use as fusion partners in the production of recombinant proteins.
As previously described affinity systems based on protein ligands are not totally suitable for industrial purposes in view of the harsh conditions required in the cleaning of columns. Therefore, there is a need for fusion partners having specific affinity towards simple and cheap organic ligands. Panning of phage display libraries of different bacterial receptors against such ligands provide novel affinity tails suitable for the use as fusion partners for the production purification of recombinant proteins.
The present invention provides means for producing and selecting proteins with novel functions. According to the invention this is achieved by extensive mutation of defined residues of stable domains of bacterial receptors. Due to the novel functions of the artificial bacterial receptors, these can be used as specific binders for therapeutic, diagnostic, biotechnology or in research.